|
| Status |
Public on Aug 25, 2009 |
| Title |
Rik1 ChIP in wild-type cells |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Rik1 ChIP in wild-type cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
cells for ip: rik1-13xmyc cells
genotype: wild type
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at 18˚C and chromatin proteins crosslinked by treatment with 10mM dimethyl adipimidate (crosslinking step was carried out only for Rik1 ChIP).
|
| Growth protocol |
Standard conditions were used to produce logarithmically growing cultures in rich media (YEA) at 30˚C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated by appropriate antibody: H2A.Z-FLAG (M2; Sigma), Pol II (8WG16; Covance), and Rik1-Myc (A14: Santa Cruz). Immunoprecipitated DNA was recovered by incubation with protein A or G slurry and reversed-crosslinked at 65˚C.
|
| Label |
Cy5
|
| Label protocol |
ChIP and whole-cell extract DNA was amplified by random-primed PCR and conjugated with Cy5 (ChIP DNA) or Cy3 (whole-cell extract DNA).
|
| |
|
| Channel 2 |
| Source name |
input DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
cells for ip: rik1-13xmyc cells
genotype: wild type
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at 18˚C and chromatin proteins crosslinked by treatment with 10mM dimethyl adipimidate (crosslinking step was carried out only for Rik1 ChIP).
|
| Growth protocol |
Standard conditions were used to produce logarithmically growing cultures in rich media (YEA) at 30˚C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated by appropriate antibody: H2A.Z-FLAG (M2; Sigma), Pol II (8WG16; Covance), and Rik1-Myc (A14: Santa Cruz). Immunoprecipitated DNA was recovered by incubation with protein A or G slurry and reversed-crosslinked at 65˚C.
|
| Label |
Cy3
|
| Label protocol |
ChIP and whole-cell extract DNA was amplified by random-primed PCR and conjugated with Cy5 (ChIP DNA) or Cy3 (whole-cell extract DNA).
|
| |
|
| |
| Hybridization protocol |
Equal amounts of Cy5-labeled ChIP DNA and Cy3-labeled whole-cell extract DNA were mixed and combined with human Cot1 DNA, Agilent Blocking Agent and Agilent Hybridization buffer, and hybridized to high-density microarrays in Agilent SureHyb hybridization chamber for 24 hours at 65˚C, 10 rpm. After hybridization, slides were washed according to Agilent protocol.
|
| Scan protocol |
Scanned on an Agilent G2505B scanner.
|
| Description |
n/a
|
| Data processing |
Data were extracted using Agilent Feature Extraction Software (CHIP-v1_95_May07 protocol). Signal was normalized by combined rank consistency filtering with LOWES intensity normalization. Background signal was estimated as a median processed signal of 152 oligonucleotides with no homology to S. pombe genome.
|
| |
|
| Submission date |
Jul 23, 2009 |
| Last update date |
Aug 25, 2009 |
| Contact name |
Shiv Grewal |
| Phone |
2407607553
|
| Organization name |
NCI
|
| Department |
LBMB
|
| Lab |
Shiv Grewal
|
| Street address |
NCI bldg 37 Rm 6068 9000 Rockville Pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL8908 |
| Series (1) |
| GSE17271 |
Histone H2A.Z cooperates with RNAi and heterochromatin factors to suppress antisense RNAs |
|
